The emergence of metabolomics promises to revolutionize the understanding of biological systems from a metabolite perspective with important implications for the diagnosis and treatment of disease. Despite significant progress in the profiling and quantitation of complex metabolomics mixtures, such as urine, serum, and cancer tissue, by NMR and mass spectrometry, important methodological challenges remain. Some of these challenges are addressed in this proposal, which is divided into four different focus areas. Specifically, it is proposed to (i) develop new approaches to integrate NMR with mass spectrometry for the de novo characterization of unknown metabolites and the more accurate identification of catalogued compounds, (ii) develop and maintain advanced web servers and databases for the reliable, efficient, and user-friendly analysis of metabolomics spectroscopic data, (iii) develop and apply new NMR experiments, and (iv) develop new approaches for sample preparation, including the use of nanoparticles for the simpler and more accurate analysis of a wide range of metabolomics samples. The proposed research will promote the dissemination of metabolomics as a powerful, versatile, and manageable tool to the biomedical scientific community. These activities are expected to lead to a better understanding of a broad range of biological questions from a metabolite perspective for the benefit of human health.